Project Summary/Abstract The central dogma of molecular biology provides an account of the mechanisms by which genetic information in the form of DNA is accurately and reliably decoded into a sequence of amino acids forming the basis of virtually all cellular processes. The primary mechanisms involved in the central dogma are transcription; the transfer of information from DNA to RNA, and translation; the synthesis of a polypeptide based on the information in messenger RNA (mRNA). Transfer RNA (tRNA) play a key role in translation, providing correct amino acids to the ribosome for addition to the nascent polypeptide by specific base-pairing with a nucleotide triplet in the mRNA sequence. The rate of translation is therefore dependent (in addition to other factors) on the availability of specific, charged (amino acid carrying) tRNAs. Thus, regulation of tRNA abundance and/or charging represent ideal points of regulation of protein synthesis, a conserved, fundamental genetic mechanism. In addition to the canonical function of tRNA in translation, these small RNA molecules have been demonstrated to regulate other processes through distinct mechanisms. Significantly, the importance of dynamic regulation of tRNAs is currently an area of emerging interest in human pathology and cancer biology. Previous findings have indicated that in response to pathogen infection, plants rapidly accumulate high levels of specific tRNAs. Such changes can be detected by traditional hybridization methods, but until very recently sequencing approaches could not be used for analysis of genome-wide changes in tRNA abundance. The high level of resolution needed to accurately distinguish individual tRNAs, which often vary by only a few nucleotides, could not be attained due to the high levels of posttranscriptional modification and complex secondary structure of mature tRNA molecules. In just the past year, a novel approach applied in mammalian systems has successfully overcome these problems. This has presented the opportunity to adapt these methods in order to perform the first ever high resolution analysis of a plant ?tRNAome? as a means of further exploring the function of differential accumulation of specific tRNAs in plant immune responses. The abundance and charging of tRNA during the induced immune response in Arabidopsis will be examined in this study through a combination of biochemical and next-generation sequencing techniques. This will be carried out in conjunction with complementary genome-wide transcriptional and translational studies currently underway in the laboratory, allowing for comparative analysis of output from multiple genetic mechanisms. In parallel, this study will characterize the functional role of a novel regulator of plant immunity that was identified in a mutant screen for translational de-regulation and has potential links to tRNA function. This work is anticipated to provide a comprehensive analysis of the dynamics and function of tRNA in rapid plant immune response.